To answer this question, I got help from instructions of Kimdar Sherefa Kemal
Materials for HIV-1 Viral RNA Isolation:
1. RNAgents R Total RNA Isolation System kit (Promega). The kit includes combined guanidine thiocyanate crystals and the Citrate/Sarcosine/β-Mercaptoethanol (CSB) buffer in a single bottle of denaturing solution. Phenol/chloroform/ isoamyl alcohol solution, isopropanol, 2 M sodium acetate (pH 4.0), and nuclease free water are also included.
2. Yeast t-RNA (Sigma).
3. Glycogen (Sigma).
4. Siliconized Flat Top microfuge tubes (Fisher Scientific).
RNA Extractions:
1. Clarify plasma by spinning at 400 × g, at room temperature, for 10 min.
2. Transfer the plasma to sterile, siliconized, 1.5 mL microfuge tubes.
3. To pellet virions, centrifuge the plasma 18, 500 × g, at 10 ◦C, for 90 min.
4. Remove most of the now virion-free plasma without disturbing the pellet.
5. To extract virion-associated RNA, re-suspend the pellet in 300 μL pre-chilled denaturing solution.
6. Vortex the pellet until it is completely dissolved. This usually takes 5–10 min.
7. Mix in order: 1 μL yeast tRNA (1 μg/μL), 1 μL glycogen (2 μg/μL) and 30 μL of 2 M sodium acetate, pH 4.0.
8. Add 32 μL of the above mixture into each tube, and then add 0.5 mL phenol/chloroform/isoamyl alcohol solution, being careful to remove only from the lower organic phase.
9. Vortex for 10 s, then chill on ice for 20 min.
10. Centrifuge at 18, 500 × g for 20 min, at 4 ◦C.
11. Transfer the top phase, which contains the RNA, to fresh microfuge tubes.
12. To precipitate the RNA, add an equal volume of isopropanol (0.3–0.5 mL) and keep the tubes at -20 ◦C for at least 2 h.
After 2 h or the Next Day
1. Pellet the RNA by centrifugation at 18, 500 × g for 30 min at 4 ◦C. Discard the supernatant.
2. Re-suspend the RNA pellet in 200 μL denaturing solution. Vortex until RNA is dissolved; usually 1 min is enough.
3. Add 300 μL isopropanol and precipitate the RNA at -20 ◦C for at least 2 h.
4. After 2 h, pellet the RNA by centrifugation at 18, 500 × g for 25 min at 4 ◦C.
5. Wash the pellet, once with 1 mL and then with 0.5 mL of 75% ice-cold ethanol, by centrifugation at 18, 500 × g for 20 min at 4 ◦C each time. During each wash break up the pellet by using RNase-free pipette tips before each centrifugation; do not vortex.
6. Air-dry the pellet for 5–20 min, depending on the amount of ethanol left in the tubes or the size of the pellet.
7. Dissolve the RNA in 25 μL nuclease-free water by using the pipette tips. Do not vortex.
8. Store the RNA at -20 ◦C until use. For long-term use store at -80 ◦C.
Hello,
I routinely extract viral RNA from cell culture supernatants and plasma using the QIAamp Ultrasens Virus Kit. It works well for isolating RNA for downstream cDNA library preps. Alternatively, I suppose you could use Trizol reagent or Invitrogen Purelink columns for total RNA but from experience the QIAamp kit works well and is quick and easy. Good luck!
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